Intercellular signaling during terminal differentiation

描述(申请人提供)：盘基网柄菌中，前孢子和前柄细胞之间的连通协调了末端分化。这些信号包括一个34个氨基酸的肽SDF-2，它的序列与神经肽DBI(安定结合抑制物)的序列有关。神经递质GABA也通过与哺乳动物的GABAB受体类似的G蛋白偶联受体作用于Dictyostelium，作为信号。另一方面，SDF-2受体DhkA是一种与DBI受体、GABAA和“外周型”BZ受体完全无关的“双组分”组氨酸激酶。细胞间的信号似乎在长时间的进化过程中一直是保守的，而受体在某些情况下发生了变化。我们将进一步阐明这一遗传易控系统中细胞间信号传递的详细机制，因为这些结果似乎与哺乳动物中枢神经系统中的信号传递有关，并可能揭示神经精神障碍的潜在机制。在前孢子和前柄细胞分别准备囊化和液泡化时，它们之间存在着复杂的信息交换。SDF-2是由前孢子细胞释放的一种蛋白质ACBA进行蛋白质分解处理而产生的。在低水平的SDF-2或GABA引发后，在前柄细胞的表面发生加工。前孢子细胞通过从囊泡中快速释放ACBA来对启动作出反应。ACBA是一种酰基-辅酶A结合蛋白，是DBI的前体。利用定点突变，我们将确定这种活性是否对内体中ACBA的积累和随后的释放是必不可少的。我们将确定对低水平的SDF-2或GABA的胞吐反应是否依赖于cAMP依赖的蛋白激酶PKA的信号。胞外ACBA被膜包埋的前柄特异性蛋白酶TAGC降解，只有在低水平的SDF-2或GABA引发后才能暴露。我们将确定这一过程是否也依赖于PKA活动。我们的结果表明，哺乳动物神经肽DBI的前体ACBP可能在一种细胞类型中合成，储存在小泡中，并由另一种细胞类型加工成活性多肽。这种相互作用可以调节这种天然配体的水平，这种天然配体与广泛使用的药物安定(安定)的靶标结合。我们的观察发现，GABA似乎可以诱导快速胞吐，这不仅解释了它在Dictyostelius中的启动活性，而且为更好地理解控制胞吐的基本过程提供了一个测试系统。总而言之，这些研究将为改善神经疾病的诊断和治疗奠定基础。

DESCRIPTION (provided by applicant): Communication between prespore and prestalk cells coordinates terminal differentiation in Dictyostelium discoideum. The signals include a 34 amino acid peptide, SDF-2, that has a sequence related to that of the neuropeptide DBI (Diazepam Binding Inhibitor). The neurotransmitter GABA is also used as a signal in Dictyostelium by acting through a G-protein coupled receptor similar to the GABAB receptor of mammals. The SDF-2 receptor, DhkA, on the other hand, is a "two-component" histidine kinase completely unrelated to the DBI receptors, GABAA and "peripheral type" BZ receptors. It seems that intercellular signals have been conserved over long periods of evolution while the receptors have changed in some cases. We will further elucidate the detailed mechanisms of intercellular signaling in this genetically tractable system since the results appear to have bearing on signaling in the mammalian central nervous system and may shed light on mechanisms underlying neuropsychiatric disorders. There is a complicated interchange of information between prespore and prestalk cells as they prepare to encapsulate and vacuolize, respectively. SDF-2 is generated by proteolytic processing of a protein, AcbA, released from prespore cells. Processing occurs on the surface of prestalk cells after priming by low levels of SDF-2 or by GABA. Prespore cells respond to priming by rapid release of AcbA from vesicles. AcbA is an acyl-CoA binding protein as is the precursor of DBI. Using site-directed mutagenesis we will determine whether this activity is essential for accumulation of AcbA in endosomes and subsequent release. We will determine whether exocytosis in response to either low levels of SDF-2 or GABA is dependent on signaling through the cAMP dependent protein kinase PKA. Extracellular AcbA is proteolytically cleaved by the membrane embedded prestalk specific protease TagC, which is only exposed following priming by either low levels of SDF-2 or GABA. We will determine whether this process is also dependent on PKA activity. Our results suggest that the precursor of the mammalian neuropeptide DBI, ACBP, may be synthesized in one cell type, stored in vesicles, and processed to the active peptides by another cell type. Such interactions could modulate the levels of this natural ligand that binds at the target of the widely used drug diazepam (Valium). Our observations that GABA appears to induces rapid exocytosis not only accounts for its priming activity in Dictyostelium but provides a test system for better understanding of the basic processes controlling exocytosis. Together these studies will add to the foundation for improvements in the diagnosis and treatment of neurological diseases.